基于AMPK调节线粒体功能探讨补肾益气方干预哮喘气道重塑的机制研究

The airway remodeling, which follows the development of airway inflammation, is the crucial point in treating asthma due to its complicacy. We have demonstrated in our previous studies that Bu-Shen-Yi-Qi formula is efficacious in treating asthma. However, its detailed mechanism remains unknown. Mitochondria has played an important role in regulating the airway inflammation and remodeling, and 5' adenosine monophosphate-activated protein kinase (AMPK) functions as a regulatory molecule for airway smooth muscle cells (ASMCs). Based on our previous work, we hypothesize that Bu-Shen-Yi-Qi formula and its active constituents might inhibit the proliferation of ASMCs and induce them apoptosis by regulating AMPK and mitochondria so as to slow the progression of the airway remodeling in asthma. Now we aim to investigate the function of Bu-Shen-Yi-Qi formula and its active constituents on the airway remodeling and mitochondria as well as its detailed regulation mediated by AMPK and mitochondria in vivo and vitro via RNAi and gene knockout. By analyzing the effect of AMPK and mitochondria abnormality on ASMCs and airway remodeling, we would elucidate their functions on ASMCs in airway remodeling and the mechanism of Bu-Shen-Yi-Qi formula and its active constituents in treating asthma. Thus, we would unveil the advantages of Bu-Shen-Yi-Qi formula and its active constituents and subsequently add more to the modern scientific connotation of Bu-Shen-Yi-Qi therapeutic method (invigorate kidney and strengthen vital Qi) from the aspect of mitochondria.

气道重塑是哮喘气道炎症发展必然结果，机制复杂，是治疗中遇到的重大挑战。团队前期研究证实，补肾益气方干预哮喘疗效确切，然而具体机制仍不明确。线粒体对哮喘气道炎症和重塑具有调节作用。AMPK分子参与调节线粒体和气道重塑中气道平滑肌细胞（ASMC）的功能。结合前期工作，我们推测补肾益气方及其活性组分可能通过调节AMPK活性和线粒体功能，抑制ASMC增殖和促进增殖的ASMC凋亡，进而延缓和拮抗气道重塑进程。本项目拟利用RNAi和基因敲除等技术手段，分别在动物整体层面和ASMC层面研究补肾益气方及其活性组分对哮喘气道重塑与线粒体的作用及基于AMPK和线粒体的机制，分析线粒体和AMPK异常对ASMC功能和气道重塑的影响，阐明线粒体AMPK在哮喘气道重塑进程中对ASMC的作用和方与组分干预的相关机制，明确补肾益气方及活性组分干预的主要环节，从线粒体功能角度丰富补肾益气治法的现代科学内涵。

气道炎症与气道重塑是支气管哮喘发生的关键病理基础。气道重塑是慢性气道炎症迁延反复所致的结果，AMPK和线粒体在调节哮喘气道炎症与重塑中发挥重要作用。补肾益气方及其主要活性成分对慢性哮喘具有一定的保护作用。本研究结果提示：（1）补肾益气方及其主要活性成分毛蕊花糖苷、芒柄花素、益母草苷对哮喘小鼠模型具有抗气道高反应性的作用。（2）补肾益气方及其主要活性成分毛蕊花糖苷、芒柄花素、益母草苷对哮喘小鼠模型具有抗气道炎症和气道重塑的作用：①能显著抑制气道周围炎症细胞浸润；②能显著抑制气道杯状细胞化生、粘液分泌、胶原沉积、平滑肌层增生肥大；③能显著抑制BALF中致炎、致重塑相关因子的表达。（3）补肾益气方对哮喘小鼠模型具有保护线粒体正常形态与功能，维持氧化/抗氧化平衡的作用：①能明显逆转哮喘小鼠肺组织线粒体损伤；②能明显恢复哮喘小鼠肺组织线粒体ATP产量；③显著降低肺组织氧化应激反应。（4）补肾益气方主要活性成分毛蕊花糖苷、芒柄花素对哮喘小鼠模型具有激活AMPK的作用。（5）补肾益气方主要活性成分毛蕊花糖苷在体外对上皮细胞具有保护作用：①抑制TNF-α诱导的Beas-2B细胞表达促炎因子；②抑制TNF-α诱导的Beas-2B细胞氧化应激损伤。（6）补肾益气方主要活性成分芒柄花素在体外对“增殖”“迁移”型支气管平滑肌细胞具有抑制作用：①抑制PDGF-BB诱导的支气管平滑肌细胞增殖；②抑制PDGF-BB诱导的支气管平滑肌细胞迁移。本项目从体内和体外实验多个方面阐述补肾益气方及其主要活性成分拮抗气道炎症与重塑的主要环节与机制，为补肾益气方防治哮喘提供了新的实验证据。